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Home , Hypoxanthine, Uric acid, Uridine

003 1 -3998/93/3406-0767$03.00/0 PEDIATRIC RESEARCH Vol. 34, No. 6, 1993 Copyright O 1993 International Pediatric Research Foundation, Inc. Printed in U.S. A.

Hypoxanthine, , and Uric Concentrations in Plasma, Cerebrospinal Fluid, Vitreous Humor, and in Piglets Subjected to Intermittent Versus Continuous Hypoxemia

LAURITZ STOLTENBERG, TERJE ROOTWELT, STEPHANIE BYASAETER, TORLEIV 0. ROGNUM, AND OLA D. SAUGSTAD Department of Pediatric Research [L.S.. T.R..S.0.. O.D.S.].Institute ofSurgica1 Research [L.S.. T.R.],and Insritute of Forensic Medicine [L.S., T.O.R.],The National Hospital, 0027 Oslo 1, Norway

ABSTRACT. Infants with sudden infant death syndrome compared with 20% in infants that die suddenly or unexpectedly have higher (Hx) concentrations in their from other causes (4). It was concluded in this study that SIDS, vitreous humor than infants with respiratory distress syn- in most cases, is probably not a sudden event but may be drome and other infant control populations. However, pre- preceded by a relatively long period of respiratory failure and vious research on piglets and pigs applying continuous . However, previous experiments on piglets applying hypoxemia has not been able to reproduce the concentra- different degrees and durations of CH have not been able to tions observed in infants with sudden infant death syn- reproduce equally high vitreous humor Hx values as observed in drome. To test whether intermittent hypoxemia could, in SIDS (5). Perhaps the higher Hx concentration formed in vitre- part, explain this observed difference, Hx, xanthine (X), ous humor of SIDS victims is due to the fact that these infants and were measured in vitreous humor, urine, suffer repeated episodes of hypoxemia before they die. In the plasma, and cerebrospinal fluid in newborn piglets during present study, we wanted to determine whether IH or CH gives intermittent hypoxemia (IH) or continuous hypoxemia higher Hx concentrations. Hx has been shown to increase in (CH) of equal degree and duration. Urinary Hx hypoxemia in other body fluids like plasma, urine, and CSF (5- was significantly higher (p < 0.04) in the IH group after 8). Concentrations of Hx, X, and Ua in plasma, CSF, urine, and 60 min of hypoxemia. The vitreous humor Hx increase was vitreous humor were measured in two groups of pigs subjected significantly higher in the IH group (from 21.0 f 7.8 to to equal periods of hypoxemia either induced intermittently or 44.1 f 25.5 pmol/L, p c 0.01 versus baseline) than in the continuously. CH group (from 16.4 f 4.2 to 23.2 f 7.3 pmol/L, p < 0.05 versus baseline) (p < 0.05 IH versus CH). X increased MATERIALS AND METHODS significantly more (p c 0.05) in vitreous humor in the IH group than in the CH group. No differences between the Approval. The experimental protocol was approved by the two groups were found in plasma and cerebrospinal fluid hospital's ethics committee for animal studies. for either Hx, X, or uric acid. We conclude that vitreous Animalpreparations. Newborn piglets (2-5 d old, weight range humor Hx and X increases more during IH than during 1530-2550 g, 18 male and eight female) were delivered by a CH. (Pediatr Res 34: 767-771,1993) local farmer on the day of the experiments. They are, from a CNS maturation point of view, close to infants of 38 wk Abbreviations gestation based on histologic findings in the CNS (9). They were premedicated with azaperone (4 mg/kg intramuscularly), can- Hx, hypoxanthine nulated in a peripheral ear vein, and given atropine (0.025 mg/ X, xanthine kg i.v.) and metomidate (4-10 mg/kg i.v.) until adequate anes- Ua, uric acid thesia was achieved. Muscle relaxation was then induced with CSF, cerebrospinal fluid pancuronium bromide (0.2 mg/kg i.v.). Additional metomidate SIDS, sudden infant death syndrome (4 mglkg) was given when necessary. Pancuronium was not IH, intermittent hypoxemia readministered. The pigs were intubated orally with a 3.0-mm CH, continuous hypoxemia cuffed Sheridan endotracheal tube (Sheridan Catheter Corp., Argyle, NY). The tube was connected via a small humidifier (Portex Termovent 600, Portex Limited, Hythe Kent, UK) to a Servo 900 B ventilator (Elema-Sch~nander,Stockholm, Sweden). The pigs were mechanically ventilated throughout the experi- ment at 40 breathslmin. ~idalvolume was adTjusted whennec- We have collected vitreous humor samples from different essary throughout the experiments to maintain arterial CO* groups of pediatric patients for several Years. These samples have between 4.6 and 5.9 kPa. Before hypoxemia, the pigs were been analyzed with regard to the metabolites Hx, X, and ventilated with room air, and only pigs with arterial oztension to some extent Ua. Hx, a well-established indicator of hypoxia ,9.3 kpa were included in the experiment. ~h~ rectal tempera- (1-319 has been found be in 80% SIDS victims ture was monitored continuously and kept between 38.0 and Correspondence and reprint requests: Dr. Lauritz Stoltenberg, Department of 39.5OC with a heating blanket and whe; necessary a heating Pediatric Research, University of Oslo, The National Hospital. 0027 Oslo 1, lamp. An arterial catheter was inserted proximally from a super- Norway. Received July 27, 1993.; accepted December 1, 1992 ficial artery on the inside of the hind leg so that ischemia to the supported by ~ri~~~td.and The Nowegian Women's Public Health A-ia- leg Was avoided. The catheter Was connected to a strain-gauge tion. L.S. is a fellow with The Nonvegian Research Council. transducer, and blood pressure was recorded continuously on a 767 768 STOLTENBERG ET AL. Gould 2600s recorder (Gould Inc. Recording Systems, Cleve- Paired or unpaired t tests with Bonferroni corrections were then land, OH). The pulse was monitored continuously via performed. Analyses were performed with an SPCC/PC+ statis- electrodes. A midline suprapubic laparotomy was performed, a tical package (SPSS Inc., Chicago, IL). A two-sided p value < urinary catheter was placed in the bladder, and a lumbar punc- 0.05 was considered significant. ture needle was placed in the cisterna magna. The pigs were placed on their side and allowed to stabilize for 30 min. During RESULTS the experiments, they received via the ear vein an infusion of a solution containing 0.7% NaCl and 1.25% at a rate of Mean survival time of the IH group including reoxygenation 10 mL/kg/h. Blood glucose was measured regularly with a re- time was 126 rnin (86 min of hypoxemia); the range was 89- 168 flectometer (Hypocount MX B, Boehringer, Mannheim, Ger- min (59- 1 18 min of hypoxemia). The mean survival time of the many), and the infusion altered as necessary to try to maintain CH group was 82 rnin (range 57-109 min). glucose between 4 and 10 mmol/L. Blood gases and mean arterial blood pressure. Table 1 shows Experimental protocol. The piglets were assigned to CH (n = the arterial O2 tension, base excess, and mean arterial blood 13) or IH (n = 13) groups. Animals in the CH group were pressure in the two groups of pigs before and after 20, 40, and ventilated with 8% O2 in , whereas animals in the IH 60 min of hypoxemia. No significant differences were observed group were given 8% 02in nitrogen for 10 rnin and 2 1 % between the two groups. However, each 5-min reoxygenation for 5 rnin repeatedly. The O2 content of the inspired air was interval in the IH group produced a significant increase in mean monitored with a Dameca OM 832 oxygen monitor (Dameca arterial blood pressure (p< 0.001). The numbers are presented Inc., Copenhagen, Denmark). in Figure 1. Blood samples. Arterial blood samples were taken after surgery Hx, X, and Ua in vitreous humor. The concentrations of Hx (-30 rnin), before hypoxemia, and then every 10 rnin in the CH in the vitreous humor of the two groups after 60 min of hypox- group and every 10 and 5 min in the IH group. Temperature- emia are given in Figure 2A. The increase was significantly higher corrected blood gases were measured with an AVL 945 Auto- in the IH group than in the CH group (p < 0.05). X also matic Blood Gas System (AVL Biomedical Instruments, increased significantly more in the IH group (1.1 f 0.2 to 3.1 f Schaffhausen, Switzerland). Blood for Hx analysis was collected 1.7 pmol/L) than in the CH group ( 1.0 f 0.1 to 1.3 + 0.4 pmol/ into prechilled EDTA tubes and centrifuged rapidly for 10 rnin L) (p < 0.05). Ua remained fairly stable. Mean values increased at 1800 X g, 4'C. Plasma was transferred to polypropylene tubes from 8.3 f 2.9 to 8.7 + 2.5 pmol/L in the IH group and from and frozen at -20°C until analysis. The withdrawn blood was 8.4 f 5.7 to 10.8 rt 5.5 pmol/L in the CH group. replaced with an equal volume of sterile NaCl (0.9%) and the Hx and X in urine. The output of Hx and X in the urine are catheter flushed with heparinized saline (4 U/mL). given in Figure 28. The Hx increase in the IH group was Sampling of CSF. Two hundred to 400 pL of CSF were drawn significantly higher (p < 0.04) than that in the CH group. No from the lumbar puncture needle before hypoxemia and then significant difference in X output was observed. after 20, 40, and 60 min of hypoxemia. CSF was collected into polypropylene tubes as long as the needle was patent and then Table 1. Blood gases, BE, and MABP (kPa) before and during frozen at -20°C for later analysis. IH (n = 13) and CH (n = 13/* Sampling of urine. The first urine sample was collected 30 min Before 20 min 40 rnin 60 rnin after surgery, and then samples were collected after every two hypoxemia hypoxemia hypoxemia hypoxemia periods of hypoxemia in the IH group and every 20 min in the CH group. Diuresis was measured and a 1-mL sample was frozen Pa02 for later measurement of Hx and X. The concentration was IH 11.9 + 1.3 3.7 + 0.5 4.2 + 0.6 4.6 + 0.8 related to diuresis, and the amount excreted per min per kg of CH 11.0 + 1.9 3.6 + 0.6 3.9 + 0.6 4.3 + 1.3 body weight was calculated. BE Sampling of vitreous humor. The conjunctiva was dissected IH -1.1 + 3.0 -9.5 + 4.0 -16.0 + 3.8 -21.0 * 5.4 free from the sclera. Vitreous humor was sampled with an empty CH -0.9 + 3.9 -10.0 + 4.3 -18.8 + 5.1 -25.2 + 5.4 Vacutainer (Vacutainer Systems Europe B.P., Maymeylan MABP Cedex, France) through a puncture of the sclera about 6 mm IH 9.8 + 1.1 5.2 + 1.3 3.6 + 0.9 2.8 It 0.9 posterior to the corneal limbus. Samples were taken only once CH 10.1 + 1.2 4.8 + 0.8 4.3 * 0.8 2.8 + 0.9 from each eye before and after 60 min of hypoxemia. One Values are given as mean + SD. Pao~,arterial 02tension; BE, base hundred to 300 pL of fluid were taken at each sampling. The excess; MABP, mean arterial blood pressure. samples were immediately centrifuged for 10 rnin at 1800 x g, 4'C, to remove cells or other contaminating particles (pigmented pieces of retina). The clear vitreous humor was transferred into polypropylene tubes and frozen at -20°C until analysis. Analysis of Hx, X, and Ua. The Hx and X levels in plasma, CSF, vitreous humor, and urine were analyzed using an HPLC method (10, 11) with modifications as previously described (5, 12). The coefficient of variation of the assay method was 0.75% based on seven separate analyses of the same sample throughout 1 d. The Ua was analyzed using an MA 100/Uric Acid UV kit from Roche Diagnostic Systems in a COBAS BIO automated blood analyzer (Hoffmann-La Roche & Co., Ltd., Basel, Swit- zerland). Each sample was analyzed three times, and the mean values were used. The standard is Seronorm, batch 181 (Ny- comed Pharma, Oslo, Norway). Statistics. Values are given as mean f SD. For vitreous humor and urine values, comparisons between groups were done with the Mann-Whitney U test and Wilcoxon matched pairs test as appropriate. For plasma and CSF values, statistical analyses were performed with two-way analysis of variance for repeated meas- Fig. 1. Blood pressure (MABP) fluctuations in the IH group. End of ures to determine the effects of time and grouptime interaction. each hypoxemia interval is indicated by HI-H6 #, p < 0.00 1. HYPOXANTHINE IN INTERMITTENT VERSUS CHRONIC HYPOXIA

0- 0- 0 20 40 60

Tme (Mln of hyporemo)

-40 -20 0 20 40 60 80 loo Lost sample Time (Min of hypoxemio)

Fig. 3. The entire set of plasma samples in six piglets from the IH group. The fluctuations in the Hx and X increases can be observed with reference to the hypoxemic (10-min) and the reoxygenation (5-min) periods.

Hx, X, and Ua in plasma and CSF. The concentrations of Hx and X are given in Figure 20and C,respectively. No significant differences could be demonstrated between the groups in either plasma or CSF. The entire set of plasma samples were analyzed K -20 o 20 40 60 Lost rampla from six piglets in the IH group and demonstrated the mode of Time (Min of hypoxamio) Hx increase in plasma (Fig. 3). Plasma Ua increased from 48.4 + 11.2 to 74.5 + 16.3 pmoll L in the IH group (p < 0.01 versus baseline) and from 44.2 + 5.8 to 65.5 & 12.9 pmol/L in the CH group (p < 0.01 versus baseline). In CSF, an increase from 3.0 + 2.2 to 9.0 + 5.9 pmoll L in the IH group and from 3.3 + I. 1 to 5.9 + 5.1 pmol/L in the CH group was observed. No significant differences could be demonstrated between the two groups for any metabolite in either plasma or CSF.

DISCUSSION One rationale for using piglets in research involving hypoxemia and is that X oxidase is present in the lungs of rats, dogs, cats, and sheep but not of and pigs (13).

-20 0 20 40 60 Lasl aompla When X oxidase is present in the lungs, Hx is cleared from the Time (Mtn of hypoxernio) circulation even during severe hypoxemia. Furthermore, a good correlation between Hx, lactate, base deficit, and pH has been demonstrated in pigs (8, 14). On the other hand, in pigs as in most other , Ua is not the end product of purine metabolism as it is in humans. For this reason, lower Hx, X, and Ua values compared with those in humans might be expected in body fluids of pigs. Vitreous humor Hx increased 6.8 pmol/L in the CH group, and this was similar to the difference (4.6 pmollL) demonstrated in a study in older pigs breathing 8% oxygen during a comparable time span ( 12). The significantly higher increase in the IH group indicates a more pronounced effect on the cellular energy charge of intermittent than chronic hypoxemia. No change in vitreous humor Ua was observed during hypoxemia, reflecting the lack

0 20 40 60 Losl somple the amount excreted per min per kg body weight. No differences were Time (Min of hyparcmio) observed with regard to the urinary output of X. Hx, however, was significantly higher (p < 0.04) in the IH group compared with the CH Fig. 2. A, Vitreous humor concentrations of Hx in the IH and CH group. C, Concentrations of plasma Hx and X. #, p < 0.01 compared groups. The Hx concentration increase in the IH group was significantly with values before hypoxemia. *, p < 0.05 compared with values before higher (p < 0.05) than in the CH group. #, p < 0.01 compared with hypoxemia. D, Concentrations of CSF Hx and X. #, p < 0.0 I compared values before hypoxemia. *, p < 0.05 compared with values before with values before hypoxemia. *, p < 0.05 compared with values before hypoxemia. B, The concentration is related to diuresis and is given as hypoxemia. 770 STOLTENBERG ET AL. of X oxidase in vitreous humor and the fact that Ua penetrates olized to X and Ua by the X oxidase (8, 27), and they poorly from plasma to vitreous humor. are potentially harmful molecules, having a direct damaging Hx was probably not washed out from vitreous humor during effect on cell membranes. In cerebral ischemia and reperfusion the short intervals of reoxygenation in the IH group. Rather, the studies, increased transport of , sodium, and sucrose was opposite was observed with a possible stepwise increase in vitre- demonstrated across the blood-brain bamer at reperfusion (28). ous humor Hx. With each reoxygenation (in the IH group), blood pressure Plasma Hx more than tripled in both groups, whereas a increased significantly (p< 0.001) to more than the prehypox- doubling was observed in the CSF. The difference in X increase emic level. This blood pressure increase lasted until the next was even more pronounced with a close to 10-fold increase in hypoxemia was introduced. It is quite conceivable that this plasma values compared with a doubling in CSF. Therefore, intermittent blood pressure increase will cause increased Hx changes in CSF do not necessarily parallel those in plasma, as filtration through beds that may be increasingly dam- indicated by our results and supported by the findings of other aged during the time span of the experiment. Furthermore, CH researchers (15). One explanation for these differences observed may not damage or change the permeability of the capillary beds in the two body fluids, may be the possibility of an active to the same degree, inasmuch as the intermittent blood pressure transport system in the choroid plexus (16). The direction of changes were not experienced by the piglets in this group. transport is not known. There are, however, speculations that In conclusion, vitreous humor and urine Hx increases signifi- this system acts to remove Hx from the CSF (17). Besides, only cantly more in IH than in CH. 40-70% of the CSF production is from the choroid plexus, whereas a substantial fraction (30-60%) is derived from the brain Acknowledgment. The authors thank Brit Engebretsen for tech- (18, 19). The brain contains higher levels of Hx phos- nical assistance. phoribosyltransferase than any other tissue (19). Therefore, in the brain, Hx is rapidly recycled into purine instead REFERENCES of lost as unsalvageable . I. Saugstad OD 1988 Hypoxanthine as an indicator of hypoxia: its role in health We have to consider the renal handling of the purine metab- and disease through free production. Pediatr Res 23: 143-150 olites in an attempt to explain the observed differences between 2. Thiringer K 1983 Cord plasma hypoxanthine as a measure of fetal hypoxia. Hx and X. The renal handling of X involves four steps: glomer- Acta Paediatr Scand 72:231-237 ular filtration, tubular reabsorption, active secretion, and postse- 3. Pietz J, Guttenberg N, Gluck I 1988 Hypoxanthine: a marker for asphyxia. Obstet Gynecol 72:762-766 cretory reabsorption, whereas Hx is eliminated mainly by filtra- 4. Rognum TO. Saugstad OD. Byasaether S, Olaisen B 1988 Elevated levels of tion along with other purine bases, the first requiring a higher hypoxanthine in the vitreous humour indicate prolonged cerebral hypoxia level of energy expenditure (20-22). There is a high renal clear- in victims of sudden infant death syndrome. Pediatrics 82:615-618 ance of Hx and it correlates with the creatinine (22), 5. Poulsen JP, Byasaeter S, Sanderud J, Rognum TO, Saugstad OD 1990 Hypo- xanthine, xanthine and uric acid concentrations in the cerebrospinal fluid, whereas the renal clearance of X can exceed creatinine clearance, plasma and urine of hypoxemic pigs. 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Letller CW 1991 Blood to brain transport Frequent episodes of brief ischemia sensitize the fetal sheep brain to neuronal after newborn cerebral ischemialreperfusion injury. Proc Soc Exp Biol Med loss and induce striatal injury. Pediatr Res 33:61-65 197:268-272

- Announcement Call for Abstracts The American Pediatric Society and The Society for Pediatric Research announce the abstract deadline for the 1994 Annual Meeting (May 2-5, 1994, Washington State Convention and Trade Center) has been set as January 4, 1994. For further information, contact: APS/SPR Association Headquarters, 14 1 Northwest Point Blvd., P.O. Box 675, Elk Grove Village, IL 60009-0675, (708) 427-0205, Fax (708) 427-1305.